Neutralization activity of IgG antibody in COVID‑19‑convalescent plasma against SARS-CoV-2 variants

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the anti-SARS-CoV-2 antibody levels, anti-spike (S)-immunoglobulin G (IgG) and anti-nucleocapsid (N)-IgG, and the neutralization activity of IgG antibody in COVID‑19‑convalescent plasma against variants of SARS-CoV-2, alpha, beta, gamma, delta, kappa, omicron and R.1 strains. The study included 30 patients with clinically diagnosed COVID-19. The anti-S-IgG and anti-N-IgG levels ranged from 30.0 to 555.1 and from 10.1 to 752.6, respectively. The neutralization activity (50% inhibition concentration: IC50) for the wild-type Wuhan strain ranged from < 6.3 to 81.5 µg/ml. IgG antibodies were > 100 µg/ml in 18 of 30 (60%) subjects infected with the beta variant. The IC50 values for wild-type and beta variants correlated inversely with anti-S-IgG levels (p < 0.05), but no such correlation was noted with anti-N-IgG. IgG antibodies prevented infectivity and cytopathic effects of six different variants of concern in the cell-based assays of wild-type, alpha, gamma, delta, kappa and R.1 strains, but not that of the beta and omicron strains. IgG is considered the main neutralizing activity in the blood, although other factors may be important in other body tissues.


Results
Anti-SARS-CoV-2 antibody titers. Figure 2 shows the levels of anti-S-IgG and anti-N-IgG measured in the 30 study patients. The anti-S-IgG and -N-IgG levels ranged from 30.0 to 555.1, and from 10.1 to 752.6, respectively. Antiviral activity of IgG antibody in convalescent plasma samples. We evaluated the antiviral activity of IgG antibody in convalescent plasma against the wild-type (WT) and variants, alpha, beta, gamma, delta, kappa and R.1 strains. Table 1 shows the IC 50 of IgG antibodies against WT and variant strains in VeroE6 TMPRSS2 cells. The IC 50 varied from < 6.3 to 81.5 µg/ml for the WT, alpha, gamma, delta, kappa and R.1 variants. On the other hand, the IC 50 of IgG antibodies against the beta variant was > 100 µg/ml in 18 of 30 (60%) subjects. Figure 3 shows the mean IC 50 of all the antibodies. The mean ± SD IC 50 values for the alpha (43.68 ± 25.36 µg/ ml in QHN001, 41.54 ± 26.52 µg/ml in QK002), gamma (32.86 ± 27.14 µg/ml), delta (44.14 ± 22.52 µg/ml) and R.1 (24.18 ± 13.62 µg/ml) variants were similar to that of WT (36.25 ± 22.72 µg/ml), but the values for the beta (82.86 ± 26.68 µg/ml) and kappa (63.19 ± 24.82 µg/ml) variants in VeroE6 TMPRSS2 cells were clearly higher than WT. The mean IC 50 of beta was significantly higher than those of WT, alpha, gamma, delta, and R.1 (p < 0.0001, Kruskal-Wallis test). The post-hoc Dunn's multiple comparisons test showed statistically significant difference in mean IC 50 between beta and WT (p < 0.0001), alpha (p < 0.0001), gamma (p < 0.0001), delta (p < 0.01), and R.1 (p < 0.0001), and also in the mean IC 50 of kappa and WT (p < 0.01), gamma (p < 0.001), and R.1 (p < 0.0001).
We also evaluated the activities of anti-SARS-CoV-2 antibodies. All the four tested antibodies showed potent activities against the WT strain, however, the activities were markedly lower in most variant strains, especially mAb1414 and 2414 nullified activity, in almost all tested variants (Table 1). Table 2 shows the IC 50 of IgG antibodies 15 against WT and variants strains in HeLa hACE2-TMPRSS2 cells. Note the higher IC 50 values for the beta and omicron variants relative to the WT.   D043  D073  D084  D091  D197  D202  D210  D213  D222  D245  D251  D310  D067  D291  D293  D242  D260  D351  D379  D384  D393  D394  D318  D373  D375  D408  D390  D409  D410   www.nature.com/scientificreports/ Correlation between neutralizing activity against WT/beta variant and antibody titers. Finally, we compared the correlation between neutralizing activities against WT, beta variant and antibody titers. The IC 50 values in WT and beta correlated significantly with anti-S-IgG levels (p < 0.05), but not with anti-N-IgG levels ( Fig. 4a-d).

Discussion
Genetic variants of SARS-CoV-2, alpha, beta, gamma, delta, kappa, omicron and R.1 strains, have infected millions of people around the world 6 . We evaluated the antiviral activities of IgG antibodies against those variants obtained from convalescent plasma samples. The IgG antibodies prevented the infectivity and cytopathic effects of three different VOCs and two VOIs in the cell-based assays using various infectious variants, WT, alpha, gamma, delta, kappa, and R.1 strains, but not those of the beta and omicron strains. These results are somewhat similar to those reported on the neutralizing activities found in BNT162b2-vaccinated individuals, which demonstrated potent activities against the alpha, delta, and kappa variants in serum samples from responders, compared with relatively moderate activity against the beta strain 16,17 . As described in the Methods section, the tested plasma samples were obtained from patients who were infected with COVID-19 between June 2020 and March 2021, and thus, it is considered that most IgGs were from convalescent plasmas of non-VOCs (Nextstrain clade 20B). Therefore, it is reasonable that the results of vaccines (prepared based on the sequence of WT) and that of the convalescent plasma samples tended to be the same 7,16-18 . We evaluated the correlation between the levels of the antibodies and neutralization activities of IgG antibodies. The higher levels of anti-S-IgG tended to suppress acquisition of viral infection. Then, the anti-S-IgG levels correlated inversely with the IC 50 values for the WT www.nature.com/scientificreports/ and beta variant. In other words, anti-S-IgG antibody prevented the infectivity and cytopathic effects in SARS-CoV-2 infection. These results indicate that anti-S-IgG antibody acts directly against the RBD of SARS-CoV-2, preventing viral entry into the cells. Similar results were reported on the correlation between anti-S-IgG levels and neutralizing activity of COVID-19-convalescent plasma 19,20 . In contrast, none of the anti-N-IgG levels correlated inversely with the IC 50 values for the WT and beta variant. These results suggest that anti-N-IgG antibody is unlikely to prevent SARS-CoV-2 infection in cells. Nevertheless, one previous study reported that anti-N-IgG antibody correlated with the neutralizing efficacy of a SARS-CoV-2 pseudovirus in all randomly selected COVID-19-convalescent plasma units 21 . Further analysis of the correlation between anti-N-IgG antibody and neutralizing activity is warranted. Our study has certain limitations. First, we did not determine the variants of SARS-CoV-2 from COVID-19-convalescent patients. However, WT and the initial alpha strains were consistent with the variants that had spread in Japan during the study period 22 . Second, the small sample size is a limitation of this study as it reduces the generalizability of the findings to a larger population. Further research with a larger sample size is needed to confirm these results. In this study, the IgG was purified and used to evaluate the activity of COVID-19-convalescent plasma, and the results correlated with S-IgG in the blood. IgG is considered to be the main neutralizing activity in the blood, although other factors may be important in neutralizing the activity in the lungs, mucous membranes, and other tissues.

Materials and methods
Patients. The study subjects were 30 patients (   www.nature.com/scientificreports/ ics Committee of the NCGM approved the study (#NCGM-G-003472-02) and each patient provided a written informed consent. The study also conformed to the principles of the Declaration of Helsinki.

Cells, viruses, antibodies and isolation of IgG fractions from COVID-19-convalescent patients.
Vero-E6 TMPRSS2 cells 23 and HeLa hACE2-TMPRSS2 cells 24   www.nature.com/scientificreports/ politan Institute of Public Health, Tokyo. Each variant was confirmed to contain each variant of concern-specific amino acid substitutions before the assays conducted in the present study (vide infra). The mAb1414, mAb2414 and mAb40591, anti-SARS-CoV-2 monoclonal antibodies, were purchased from Active Motif (Carlsbad, CA) and Sino Biological (Beijing, China), respectively. The pAbA19215, anti-SARS-CoV-2 polyclonal antibody was purchased from ABclonal (Woburn, MA). Plasma or serum samples were collected from patients, and IgG fractions were purified using a spin column-based antibody purification kit (Cosmo Bio, Tokyo) according to the instructions provided by the manufacturer. Briefly, serum or plasma was collected, heat-inactivated for 30 min at 56 °C, and spin columns were centrifuged at 3500 rpm for 5 min. The IgG fractions in the supernatants were eluted and collected. Antiviral assays. The neutralizing activities of IgG fractions from COVID-19-convalescent plasma were determined by quantifying the IgG antibody suppression of the cytopathic effect (CPE) of each SARS-CoV-2 strain in VeroE6 TMPRSS2 cells and HeLa hACE2-TMPRSS2 cells, using the procedures described previously 7,13,16,17 .
Briefly, each of the purified IgG fraction was two-fold serially diluted in the culture medium. After 3-day culture of the cells, the level of cytopathic effect (CPE) observed in SARS-CoV-2-exposed cells was determined using the WST-8 assay, employing Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The IgG antibody dilution that yielded 50% inhibition of CPE was defined as the 50% Inhibition Concentration (IC 50 ). Each of the purified IgG fractions was tested in duplicate.
Statistical analysis. Data are expressed as mean ± standard deviation (SD). Differences between groups were analyzed for statistical significance using Kruskal-Wallis test. When the latter test was significant, posthoc Dunn's multiple comparisons test was applied. Correlations between two assays were analyzed for statistical significance using nonparametric Spearman test. A p value < 0.05 denoted the presence of statistically significant difference. All statistical analyses were performed using the GraphPad Prism software version 8 (GraphPad Software, San Diego, CA).
Institutional review board statement. The Ethics Committee at the NCGM approved the present study (#NCGM-G-003472-02). Each patient provided written informed consent. The study also conformed to the Declaration of Helsinki principles.

Data availability
The datasets generated during and/or analyzed during the study are available from the corresponding author on reasonable request.